Processes for making hydrazides

ABSTRACT

A method is disclosed for preparing hydrazides from hydrazine and an acyl chloride which comprises the steps of (a) preparing a stirred substantially uniform slurry comprising hydrazine and an inert solvent at low temperature; and (b) adding an acyl chloride continuously to said slurry. The method avoids or limits production of undesired bis-hydrazide by-products. The method is used to prepare 3-methyl-3-mercaptobutanoic acid hydrazide, a molecule used to link calicheamicin to a monoclonal antibody.

CROSS-REFERENCE TO RELATED APPLICATION

Priority is claimed to U.S. patent application Ser. No. 13/755,886,filed on Jan. 31, 2013, which itself claims priority to U.S. patentapplication Ser. No. 13/360,220, filed on Jan. 27, 2012, which itselfclaims priority to U.S. patent application Ser. No. 12/123,777, filed onMay 20, 2008, which itself claims priority to U.S. Prov. Patent Appl.No. 60/939,529, filed May 22, 2007, all of which are hereby incorporatedherein in entirety.

FIELD OF INVENTION

The present invention relates to an improved synthetic method forpreparing hydrazides from hydrazine and acyl chlorides. The hydrazideproducts have a protected thiol group which is used to linkcalicheamicin to monoclonal antibodies.

BACKGROUND OF THE INVENTION

MYLOTARG® (gemtuzumab ozogamicin), also referred to as CMA-676 or justCMA, consists of a monoclonal antibody against CD33 that is bound tocalicheamicin by means of an acid-hydrolyzable linker. When thederivatized calicheamicin binds to the DNA minor groove, it disrupts DNAprogression and eventually causes cancer cell death. The commercialproduct was marketed as the first antibody-targeted chemotherapeuticagent under the name MYLOTARG® and is currently approved for thetreatment of acute myeloid leukemia (AML) in elderly patients.

3-Methyl-3-mercaptobutanoic acid hydrazide, also called DMH linker, orCL-332258, is used to link calicheamicin to monoclonal antibodies. Thederivatized calicheamicin is then activated for conjugation with ahumanized monoclonal antibody to give CMA-676. Currently, DMH linker isprepared through a 5-step reaction process via the intermediate,p-methoxybenzylthioether hydrazide, 5. (Equations I-V). In the presentmanufacturing process, a Michael addition of p-methoxy-benzylthiol to3,3 dimethylacrylic acid is assisted by piperidine, (Equation I).

The resulting thioether acid (1) reacts with oxalyl chloride inmethylene chloride to form p-methoxybenzylthioether acid chloride (2)(Equation II).

Acid chloride (2) is slowly added to a mixture of hydrazine/methylenechloride (in a ratio of about 28%, v/v) at low temperature (−70° C.).The corresponding p-methoxybenzylthioether hydrazide (3) forms in about74% yield (Equation III):

However, the desired product p-methoxybenzylthioether hydrazide (3)typically contains about 20% of an undesired by-product,bis-methoxybenzylthioether hydrazide (see Equation VI below). Removingthe benzyl protecting group under acidic conditions (Equation (IV),followed by neutralization of the acid salt and purification (EquationV) provides 3-methyl-3-mercaptobutanoic acid hydrazide (5) in 45% yield.

An undesired by-product, bis-methoxybenzylthioether hydrazide (6) isgenerated from the reaction of the product p-methoxybenzylthioetherhydrazide with the starting material p-methoxybenzylthioether acidchloride (Equation VI). The generation of this by-product results inlower yield and quality.

Using original process procedures, the bis-methoxybenzylthioetherhydrazide (6), is generated in amounts of about 20%. The presence ofthis level or greater of undesired by-product from Equation III isclearly undesirable. The present invention describes techniques whichovercome this problem and decrease the yield of the undesiredby-product.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a process schematic for preparing p-MethoxybenzylthioetherAcid (1).

FIG. 2 is a process schematic for preparing p-MethoxybenzylthioetherAcid Chloride (2) and p-Methoxybenzylthioether Acid Hydrazide (3).

FIG. 3 is a process schematic for preparing DMH LINKER (5).

SUMMARY OF THE INVENTION

Certain embodiments of the present invention provide processes forsynthesizing hydrazides (e.g., 3-methyl-3-mercaptobutanoic acidhydrazide (4)) while reducing the level of the by-productbis-methoxybenzylthioether hydrazide (e.g., (6)) from about 20% to about3 to 5%. In an embodiment of the invention, a methoxybenzylthioetheracid chloride (2) solution is added to a stirred hydrazine/methylenechloride mixture which is more dilute than that of the original process(e.g., volume ratio=14% vs. about 24% to 32% v/v in the originalprocess). A preferred volume ratio for dilution is about 14% v/v.Optionally and without limitation other non-reactive (or inert)halogenated solvents instead of or in combination with methylenechloride may be used in the embodiments of the invention describedherein to form the hydrazine mixture to which the acid chloride isadded. Preferred examples of such other solvents include carbontetrachloride, chloroform, ethylene dichloride and chlorobenzene. Incertain embodiments, the amount of methylene chloride (or correspondinginert solvent) is doubled, significantly reducing the amount of unwantedbis-hydrazide by-product. In certain embodiments, themethoxybenzylthioether acid chloride solution is added to thehydrazine/methylene chloride slurry continuously, at a steady rate,rather than portion wise. In certain embodiments, the addition rate isadjusted to maintain a reaction temperature of −68 to −75° C. In certainembodiments, an agitation speed between 300 to 400 rpm in a round-bottomflask or 270 rpm in a Morton-type flask is used. Certain methods of theinvention have afforded p-methoxybenzylthioether hydrazide (3) in 91.1%strength in 85% yield with about 4.7% of the bis-methoxybenzylthioetherhydrazide (6) formed.

In another embodiment of the invention, it was found that despite theimprovement afforded by using the more dilute methylene chloride system,it was still necessary to scrape frozen crystallized hydrazine off thebottom and sides of the reactor vessel. The standard practice hadpreviously been to cool the methylene chloride/hydrazine solutiontogether to approximately −70° C. This resulted in a significant portionof the hydrazine crystallizing and precipitating on the sides of thevessel. To ensure that all the hydrazine was available for reaction, itwas necessary to scrape the material from the walls of the vessel toallow it to form a stirrable slurry. To avoid this situation, analternative procedure as part of certain embodiments of the presentinvention was devised. This alternative procedure involves the chillingof the methylene chloride to −68 to −75° C., preferably 70° C., followedby a slow drop wise addition of hydrazine to the cold methylene chlorideto form a uniform slurry. This new procedure achieves the formation of amuch more uniform hydrazine slurry, which minimizes crystallizedhydrazine formation on the inner walls of the flask, and reduces oreliminates the need to scrape the flask. This allows the desired amountof hydrazine to be available for reaction, which helps reduce theformation of the bis-methoxybenzylthioether hydrazide (6).

Certain embodiments of the invention provide a method involving thecontinuous addition of a methoxybenzylthioether acid chloride (2)solution to a comparatively dilute (from the perspective of the priorprocess) and stirrable chilled hydrazine/methylene chlorideheterogeneous mixture (preferably having a hydrazine concentration ofapproximately 14%). The methoxybenzylthioether acid chloride solution isadded to the hydrazine/methylene chloride slurry continuously, at anapproximately steady rate rather than portion wise. The addition rate ispreferably adjusted to maintain a reaction temperature of −68 to −75° C.An agitation speed between 300-400 rpm in a round-bottom flask or 270rpm in a Morton-type flask is preferred. The improved processes ofcertain embodiments of the invention reduce the level of the by-product,bis-methoxybenzylthioether hydrazide (6), from the previously achievedlevel of about 20% to about 3 to 5% or lower. The improved synthesis ofintermediate 1 improves the efficiency of the overall process ofsynthesizing MYLOTARG®.

Certain embodiments of the invention provide a method of preparing ahydrazide from hydrazine and an acyl chloride comprising the steps of(a) preparing a stirred substantially uniform slurry comprisinghydrazine and an inert solvent; and (b) adding an acyl chloridecontinuously to said slurry. In another aspect of this embodiment, theacyl chloride is added substantially drop wise to the slurry in theaddition step (b).

Certain embodiments of the invention provide methods of preparinghydrazides from acyl halides and hydrazines. In one embodiment, thepreparation is accomplished via a chemical reaction between anelectrophilic acyl carbonyl of the acyl chloride and a nucleophilicnitrogen of hydrazine. The particular substituents attached to the acylcarbonyl which are suitable for the invention can be any moiety whichdoes not interfere with hydrazide bond formation, including suchmoieties which incorporate protecting groups in order to preventinterference with hydrazide bond formation. For example, in certainembodiments of the invention, an acyl halide comprises a protectedthiol. Examples of protected thiols include but are not limited tobenzyl thioethers.

In another aspect of the invention, acyl chlorides have a structure:

wherein P is a thiol protecting group, R₁ and R₂ are each selected fromthe group consisting of C₁-C₅ alkyl, and L is an alkylene linker.Examples of alkylene linkers L include but are not limited to L is—(CH₂)_(n)—, where n is an integer 1 to 5. In certain embodiments, R₁and R₂ are the same, such as when R₁ and R₂ are both the same C₁-C₅alkyl. Examples of C₁-C₅ alkyl include but are not limited to methyl,ethyl, propyl, butyl, pentyl, including both linear and branched isomersthereof. Examples of thiol protecting groups include but are not limitedto a benzyl group, wherein the phenyl moiety is optionally substituted.Examples of optional substituents include but are not limited to analkoxy, such as methoxy, ethoxy and the like. Accordingly, in oneembodiment of the invention, an acyl chloride has the structure:

Another embodiment of the present invention is a method of preparing ahydrazide from hydrazine and an acyl chloride comprising a first step ofpreparing a stirred substantially uniform slurry comprising hydrazineand an inert solvent. In another aspect of this embodiment, an inertsolvent is methylene chloride.

Another embodiment of the present invention is a method of preparing ahydrazide from hydrazine and an acyl chloride. In another aspect of thisembodiment, a hydrazide product has the structure:

wherein P is a thiol protecting group. In yet another aspect of thisembodiment, P is a benzyl group, optionally substituted on the phenylring. In another aspect of this embodiment, P is p-methoxybenzyl groupand R₁ and R₂ are each selected from the group consisting of C₁-C₅ alkyland L is an alkylene linker. Examples of alkylene linkers include butare not limited to L is —CH₂—. In one embodiment, R₁ and R₂ are eachindependently methyl.

Another embodiment of the present invention are hydrazide productsprepared according to methods of the present invention. In oneembodiment, a desired hydrazide has a structure:

or a salt thereof. In yet another embodiment of the invention, thehydrazide is 3-methyl-3-mercaptobutanoic acid hydrazide.

In another aspect of the invention, the desired hydrazide productcontains less than 5% of a bis-hydrazide by-product having thestructure:

wherein R and R′ are optionally substituted alkyl, heteroalkyl, orheteroalkaryl groups.

In another aspect of this embodiment, R and R′ moieties in abis-hydrazide by-product are each

in which P is a thiol protecting group, R₁ and R₂ are each selected fromthe group consisting of C₁-C₅ alkyl, and L is an alkylene linker.Examples of alkylene linkers, L, include but are not limited to —CH₂—.In another aspect of this embodiment, R₁ and R₂ are each independentlymethyl. In another aspect of this embodiment, P is a benzyl group,optionally substituted on the phenyl ring; examples include but are notlimited to P is p-methoxybenzyl group.

Another embodiment of the invention is a method of preparing a hydrazidefrom hydrazine and an acyl chloride wherein the hydrazide productcontains less than 5% of a bis-hydrazide by-product having a structure:

Another embodiment of the present invention is a method of preparing ahydrazide from hydrazine and an acyl chloride comprising a first step ofpreparing a stirred substantially uniform slurry comprising hydrazineand an inert solvent, and second step of then adding an acyl chloridecontinuously to the slurry. In another aspect of this embodiment, thecontinuous addition of acid chloride solution is adjusted to maintainreaction temperature of about −68° C. to about −75° C. In yet anotheraspect of this embodiment, the hydrazine slurry is substantiallyuniform.

In another embodiment of the invention, a hydrazide linkage is preparedaccording to a method comprising the steps of (a) cooling a reactionvessel comprising a stirred inert solvent to a desired low temperature;(b) adding hydrazine in a continuous fashion to said reaction vesselthereby preparing a stirred substantially uniform slurry comprising thehydrazine and the inert solvent; (c) adding an acid chloride to saidslurry in a continuous fashion, thereby forming a hydrazide linkage. Inanother aspect of this embodiment, the inert solvent is methylenechloride.

In another embodiment of the invention, a hydrazine slurry is preparedby a method comprising the steps of (a) chilling an inert solvent to atemperature of about −68° C. to about −75° C., and (b) adding hydrazinedissolved in an inert solvent drop wise to the cold inert solvent. Inanother aspect of this embodiment, the inert solvent is methylenechloride. In still another aspect of this embodiment, the hydrazineslurry is stirred at a speed of about 270 to about 400 rpm.

Another embodiment of the invention is a method of preparing gemtuzumabozogamicin comprising the steps of preparing the linker3-methyl-3-mercaptobutanoic acid hydrazide and using said linker to linkcalicheamicin to the monoclonal antibody gemtuzumab.

DETAILED DESCRIPTION OF THE INVENTION

p-Methoxybenzylthiol undergoes a Michael addition with3,3-dimethylacrylic acid in piperidine. The quantities of reagentsaffect the outcome of the reaction. In one embodiment, the quantity ofp-methoxybenzyl thiol is in slight molar excess over the3,3-dimethylacrylic acid, such as the range of between 0.354 (2.3 mol)and 0.362 kg (2.35 mol). If the amount is below this range, thesubsequent reaction may be incomplete. If the amount is above thisrange, the excess reagent may complicate processing. The reactionmixture is heated, taking care not to exceed about 98° C. for a minimumof about 15 hours in order to avoid the excessive formation ofimpurities. The piperidine is removed by dilution with methylenechloride and washing with aqueous hydrochloric acid and then water.Keeping the temperature above 50° C. and less than 90° C. is necessarybefore and during the addition of HCl to avoid precipitating thereaction product. The reaction is further cooled and then extracted withmethylene chloride as directed in the experimental section.

The amounts of solvents used are proportional to the scale of thereaction for optimum results and purification. The resulting CH₂Cl₂product solution is dried with magnesium sulfate, clarified,concentrated under vacuum, then diluted with heptane to precipitate thecrude intermediate, which is filtered and washed with heptane.Purification is accomplished by redissolving the crude material inmethylene chloride and precipitating again with heptane. The purifiedp-methoxybenzylthioether acid (1) is isolated by filtration, washed withheptane and dried under vacuum.

p-Methoxybenzylthioether acid (1) is converted to the corresponding acidchloride using oxalyl chloride with methylene chloride as the solvent.Oxalyl chloride should be present in molar excess with respect to thep-methoxybenzylthioether acid for complete reaction. Acid chlorideproduct is isolated by concentration under vacuum to remove methylenechloride/excess oxalyl chloride to an oil. The resulting oil is dilutedwith methylene chloride and added slowly over time for about 3 to 5hours at a temperature range of 65 to 75° C. to a diluted mixture ofhydrazine and methylene chloride.

One aspect of the present invention is the formation of a uniform slurrycomprising hydrazine and an inert solvent such as methylene chloride.According to one embodiment of the invention, a uniform slurry isprepared by the slow drop wise addition of liquid hydrazine to methylenechloride which had been pre-chilled to about 68 to −75° C., preferably−70° C., prior to commencing the addition of hydrazine. By contrast,cooling a premixed solution of hydrazine in methylene chloride to thesame temperature results in the less favorable formation of acrystalline hydrazine which collects on the sides of the reactionvessel. Without being bound to theory, it is believed that the slow,drop wise addition of hydrazine to the pre-chilled methylene chlorideand control of the maximum concentration of hydrazine in the methylenechloride results in the formation of smaller, more uniform crystals ofhydrazine which remain suspended in the stirred mixture of methylenechloride and substantially do not freeze to the walls of the vessel.Formation of a substantially uniform slurry helps to ensure thathydrazine remains in contact with the stirred methylene chloride and isavailable for reaction with the incoming acid chloride solution.Formation of a uniform slurry obviates the need to scrape the inside ofthe reaction flask as required in the prior process. Additionally, thisensures that the desired amount of hydrazine is available for reaction,which also reduces the amount of bis-methoxybenzylthioether hydrazide(6) formed.

The concentration of hydrazine in methylene chloride affects the amountof bis-methoxybenzylthioether hydrazide (6) that is formed as aby-product. In prior processes, the concentration of hydrazine/methylenechloride was about 24 to 32% v/v. Halving the ratio ofhydrazine/methylene chloride (more dilute hydrazine) to about 12 to 16%v/v, preferably about 14% v/v, resulted in a decrease in the amount ofundesired bis-methoxybenzylthioether hydrazide (6) formed (see Table 1).

TABLE 1 PROCESS RESULTS AFTER IMPROVEMENT Corrected Strength ofBis-hydrazide Hydrazide hydrazide* by-product Yield Exp. No. (%) (%) (%)1.1 91.61 9.69 85.4 1.2 95.08 5.76 87.7 1.3 96.16 4.13 89.6 1.4 93.767.36 85.7 *as determined by high pressure liquid chromatography

In certain embodiments of the invention, the acid chloride solution isadded to the hydrazine/methylene chloride slurry continuously, at asteady rate rather than portion wise. The amount of added acid chlorideand rate of its addition both affect the yield of desiredmethoxybenzylthioether (3). If too little acid chloride is added,excessive amounts of bis-methoxybenzylthioether hydrazide (6) by-productmay form. Also, if the addition time of acid chloride is too short, lessthan the 3 hrs, excessive amounts of bis-methoxybenzylthioetherhydrazide (6) by-product may form. The addition rate is adjusted tomaintain a reaction temperature of −68 to −75° C. If the reactiontemperature rises to higher temperatures, excessive amounts of thebis-methoxybenzylthioether hydrazide (6) may also form. An agitationspeed between 300 to 400 rpm in a round-bottom flask or 270 rpm in aMorton-type flask is preferably used to stir the hydrazine slush. Bothaspects of the improved process, use of a more dilutehydrazine/methylene chloride mixture and the formation of a uniformslurry, reduce the level of the by-product, bis-methoxybenzylthioetherhydrazide (6), from about 20% to about 3 to 5%. The improved processsteps of forming the hydrazine improves the overall efficiency ofsynthesizing the linker 3-methyl-3-mercaptobutanoic acid hydrazide andtherefore also improves the overall efficiency of preparing MYLOTARG®(gemtuzumab ozogamicin).

Upon reaction completion, the reaction mixture is concentrated undervacuum and the residue is treated with methanolic sodium hydroxide(about 4 to 5%). This solution is concentrated under vacuum, dilutedwith methylene chloride, washed with water, dried over magnesiumsulfate, clarified, and concentrated under vacuum to a concentrate. Careshould be taken to use sufficient magnesium sulfate to completely drythe product so there is no decomposition or interference withcrystallization of the product in the next synthetic step. The finalconcentrate is diluted with methylene chloride in an amount of 1.33times the weight of p-methoxybenzylthioether acid (1), and this solutionis added to diethyl ether in an amount of 7.6 times the weight ofp-methoxybenzylthioether acid (1). An aliphatic hydrocarbon solvent suchas heptane, hexane, octane or iso-hexane, preferably heptane, in anamount of 1.83 times the weight of p-methoxybenzylthioether acid (1) isadded to the resulting slurry to complete the precipitation. Thep-methoxybenzylthioether hydrazide (6) is isolated by filtration, washedwith heptane, and dried under vacuum.

p-Methoxybenzylthioether hydrazide (3) is treated withtrifluoromethanesulfonic acid in the presence of anisole, usingtrifluoroacetic acid as a solvent. Care must be taken during theaddition and subsequent reaction time not to exceed a reactiontemperature of about 20° C. in order to avoid the formation of undesiredimpurities. After cleavage of the p-methoxybenzyl protecting group iscomplete, the reaction mixture is quenched into methanol and filtered toremove solid by-products. The filtrates are concentrated under vacuum,dissolved in water, washed with methylene chloride, and treated with ananionic exchange resin to give 3-methyl-3-mercaptobutanoic acidhydrazide (5). The resin is removed by filtration and then aqueoushydrochloric acid is added to the crude product solution to form the HClsalt. The batch is concentrated under vacuum, dissolved in ethanol,clarified by filtration, and concentrated under vacuum. This concentrateis diluted with ethyl acetate and concentrated under vacuum. Again, theresidue is diluted with ethyl acetate then isolated by filtration. Thewet cake is heated with ethyl acetate to about 48 to 55° C., cooled,filtered, and suction dried. The dried HCl salt is converted to the freebase by treatment with an anionic exchange resin in water. The resin isremoved by filtration and the filtrates are concentrated under vacuum.The concentrate is dissolved in ethanol, concentrated under vacuum,slurried in diethyl ether, and concentrated under vacuum. As a finalpurification, 3-methyl-3-mercaptobutanoic acid hydrazide (5) isdissolved in methylene chloride, clarified by filtration, and treatedwith silica, which is then removed by filtration. The purified productin solution is isolated by concentration under vacuum. In a preferredpurification method, demonstrated in Example 15,3-methyl-3-mercaptobutanoic acid hydrazide (5) is dissolved in 50 parts(v/w) of methylene chloride at 20° C.±3° C., stirred 30 minutes andfiltered. The resulting solution is treated with 0.7-1 part (w/w vscrude linker) silica gel, stirred 30 minutes, filtered, and concentratedto dryness on a rotary evaporator. The resulting solid is trituratedwith n-heptane. After isolation and drying in vacuo,3-methyl-3-mercaptobutanoic acid hydrazide (5) is obtained as a freeflowing solid in approximately a 76% yield.

One aspect of the present invention is a process that providesp-methoxybenzylthioether hydrazide with less than 5% of the undesiredby-product, bis-methoxybenzylthioether hydrazide (6). This improvedprocess comprises a modified method for the coupling of thioether acidchloride with hydrazine to form p-methoxybenzylthioether hydrazide. Theprocess steps are shown schematically in Equation I. The undesiredby-product, bis-methoxybenzylthioether hydrazide (6), is generated fromthe coupling of the product, p-methoxybenzylthioether hydrazide (3),with the starting material, p-methoxybenzylthioether acid chloride (2).The generation of undesired bis-methoxybenzylthiothioether hydrazide (6)results in lower quality and yield.

In another aspect of the present invention, the process disclosure maybe conceptually understood to encompass broader applications. Thespecific reaction sequence (Equation III) may be generalized in terms ofEquation VII:

Where a material is described as added continuously in a process step,such addition is meant to occur steadily for a period of time ratherthan portion wise or all at once. Drop wise addition of a liquid oraddition of a liquid through a steady stream are examples of continuousaddition. In certain embodiments, continuous addition is accomplished bycontrolling the rate of addition of a material which reacts exothermallyat a rate slow enough to maintain a reactant temperature within acertain temperature range.

Slurry as used herein refers to a combination of solid and liquid phasesthat are intimately mixed together and typically chilled to atemperature which supports the presence of both solid and liquid phaseswhereas the mixture would be purely liquid at ambient temperatures.Slurry is sometimes used to refer to a mixture of a solid/liquid mixtureof the same substance such as an ice/water mixture in which the ice isrelatively finely divided and intimately mixed with the liquid water. Inthe context of this invention, slurry can refer to a solid/liquidmixture formed from the combination of two materials such as hydrazineand a solvent such as methylene chloride. In a chilledhydrazine/methylene chloride slurry, the liquid phase is believed tocontain a mixture of methylene chloride and hydrazine while the solidphase is believed to primarily be hydrazine.

The term “alkyl” includes a straight or branched alkyl having 1 to 10carbon atoms and a lower alkyl having 1 to 5 carbon atoms is preferable.For example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl,2-methylbutyl, n-hexyl, isohexyl, heptyl, isoheptyl, octyl, isooctyl,nonyl, decyl and the like are included. The term “alkylene” includesstraight and branched diradicals of alkanes having one to 10 carbonssuch as methylene (—CH₂—), ethylene (—CH₂CH₂—), propylene, butylene, andpentylene.

The term “heteroalkyl” refers to an alkyl group as defined herein whereone or more carbon atoms are replaced by a heteroatom (an oxygen,sulfur, nitrogen, or phosphorus atom) and may optionally containadditional heteroatoms. The term heteroalkaryl refers to a heteroalkylmoiety as described above but which is further substituted with an arylmoiety wherein such aryl moiety may be optionally substituted.Optionally substituted aryl includes phenyl and substituted phenyl. Insubstituted phenyl, one, two or three optional substituents maysubstitute for hydrogen on a phenyl ring and be situated ortho, meta,and/or para to the methylene group of the benzyl carbon (or other pointof attachment). In Example 1, a methoxy group is situated para to themethylene group. Non-limiting examples of optional aryl substituentsinclude, C₁-C₅ alkyl, C₁-C₅ alkoxy, C₁-C₅ haloalkyl, C₁-C₅ haloalkoxy,wherein the hydrogen atoms attached to the alkyl or alkoxy carbons maybe replaced by halogen atoms, as for example, in —CF₃ and —OCF₃.

The following non-limiting examples illustrate the invention.

Example 1 Original Preparation of p-Methoxybenzylthioether Acid (1)

With reference to Equation I, a 5-L round bottomed flask, equipped witha thermocouple, mechanical stirrer, reflux condenser topped with N₂inlet, and 250 mL pressure equalizing addition funnel, was charged with400 g, 465 mL, 4.70 mol of piperidine. 3,3-dimethylacrylic acid (215 g,2.15 mol) was added portion wise into a stirred 5-L reaction flask. Thereaction was vigorously stirred under N₂. The reaction temperature wasmaintained to less than 35 to 40° C. during the addition (Note: strongexotherm, i.e., off gassing). p-Methoxybenzylthiol (386 g, 323 mL, 2.32mol) was charged via pressure equalizing addition funnel over 15 minutesinto the (5-L) reaction flask. The mixture was heated to 82 to 88° C.,with stirring under N₂. The reaction temperature was maintained in thisrange for 15 minutes. Note: exothermic. The clear yellow mixture washeated to 92 to 95° C. with stirring under N₂ for a minimum of 15 hours.A 1 mL sample was removed for HPLC analysis. The reaction was deemedcomplete when less than 3% by area of the 3,3-dimethylacrylic acidremained. The reaction was cooled to 70 to 75° C. by removing theheating mantle.

3M hydrochloric acid solution (1,900 mL, 2,090 g) was added via a 1-Lpressure equalizing addition funnel to the stirred yellow solution whilemaintaining the temperature to less than 90° C. The final temperaturewas 70 to 75° C. The mixture was cooled to 20 to 25° C. by applying acool water bath. Methylene chloride (1,600 g, 1,210 mL) was charged tothe heterogeneous mixture. The mixture was stirred for 5 minutes. The pHof the upper aqueous layer in the flask was checked and 3M HCL was addedas necessary until the aqueous layer had pH less than 2. The entirecontents of the 5-L reaction flask was transferred to a 4-L separatoryfunnel. The two layers were allowed to separate for a minimum of 10minutes. The organic (bottom) layer was transferred from the separatoryfunnel back into the 5-L reaction flask. The aqueous (top) layer wastransferred from the separatory funnel into a (4-L) Erlenmeyer flask. 3Mhydrochloric acid solution (1,050 mL, 1,154 g) was charged via 1,000 mLpressure equalizing addition funnel to the methylene chloride solutionin the 5-L vessel over 10 minutes. The pH of the upper aqueous layer inthe flask was checked and 3M HCL was added as necessary until theaqueous layer pH was less than 2. The entire contents of the 5-Lreaction flask was transferred to a 4-L separatory funnel. The combinedvolume was recorded. The two layers were allowed to separate for aminimum of 10 minutes. The organic (bottom) layer was transferred fromthe separatory funnel into a clean 4-L Erlenmeyer flask. The aqueous(top) layer was transferred from the separatory funnel into a clean 4-LErlenmeyer flask. The aqueous layers were combined into a 5-L reactionflask.

Methylene chloride (305 mL, 400 g) was charged into the aqueous solutionobtained in the previous step. The mixture was stirred for a minimum of5 minutes. The entire contents of the 5-L flask was transferred to a 6-Lseparatory funnel and the combined volume was recorded. The two layerswere allowed to settle for at least 5 minutes. The organic (bottom)layer was transferred from the separatory funnel into the 4-L Erlenmeyerflask. The methylene chloride solution was washed with 1,000 mL ofwater. The mixture was stirred thoroughly for 1 to 2 minutes and thenallowed to settle for a minimum of 10 minutes. The aqueous layer wasseparated. The pH of the aqueous layer in the flask was measured. Theaqueous layers were combined and discarded. Anhydrous magnesium sulfate(110 g) was added to the methylene chloride solution and stirred for atleast 15 minutes. A (5-L) reaction flask was pre-marked at 800, 900 and1,000 mL levels. Using suction, the yellow mixture was filtered througha 15 cm Buchner funnel with filter paper (Whatman #1) into a 5-Lreaction flask. The flask and filter cake were rinsed with 300 mL, 400 gof methylene chloride. The methylene chloride solution was concentratedas follows: The 5-L round-bottomed flask was equipped with a mechanicalstirrer and a Claisen still head fitted with a thermocouple. The stillhead was connected to a 30-cm simple condenser, and the condenser wasattached to a receiver adaptor which was fitted with a 1-L flask cooledin an ice bath. The receiver adaptor was connected to an ice cold trap.The cold trap was connected to a vacuum pump.

Methylene chloride from the solution in the 5-L flask was distilled at atemperature of 15 to 35° C., under vacuum, until a pot volume of about900 mL was attained. The distillate was discarded. The temperature ofthe contents of the 5-L reaction flask was adjusted to from 15 to 20° C.Heptane (2,442 mL, 1,670 g) was charged via a pressure equalizingaddition funnel to the stirred concentrate solution over a minimum ofabout 10 minutes. A precipitate formed after addition of approximately1,000 ml, 684 g heptane. The heterogeneous mixture was cooled withstirring to a temperature of from 0 to 5° C. over a minimum of 20minutes and held at that temperature for a minimum of 30 minutes. Thecontents of the 5-L reaction flask were filtered through a 30 cm Buchnerfunnel with Whatman #1 filter paper. The filtrate was collected in a 4-Lsuction flask. The 5-L reaction flask was rinsed to the filter cake with2×310 mL, 2×212 g of heptane. The filter cake was dried with suctionuntil essentially no more filtrate was collected and for a minimum of 25minutes. The filter cake height was about 20 mm. The filter cake weightwas about 601 g. The filtrate was discarded. The cake was transferredinto a 5-L round bottomed flask equipped with thermocouple, a mechanicalstirrer, an N₂ inlet and a 1-L pressure equalizing addition funnel.Methylene chloride (750 mL, 990 g) was charged into the 5-L reactionflask and stirred until all solids dissolved (about 10 minutes). Heptane(1,060 mL, 725 g) was charged into the 5-L reaction flask. Theheterogeneous solution was cooled to 0 to 5° C. using an ice bath over aminimum of 15 minutes and then stirred for at least 30 minutes. A thickheterogeneous solution was observed. The contents of the 5-L reactionflask were filtered on 30 cm Buchner funnel with Whatman #1 filterpaper. The filtrate was collected in a 4-L suction flask. The 5-Lreaction flask was rinsed to the filter cake with 2×310 mL, 2×212 gheptane. The filter cake was dried with suction until essentially nomore filtrate was collected (a minimum of 20 minutes). The filter cakeheight was about 20 mm. The filter cake weight was about 632 g. Thefiltrate was discarded. The wet cake was transferred into a drying dish.The drying dish containing the p-methoxybenzylthioether acid was coveredwith clean filter paper. The product was dried in a vacuum oven at 38 to40° C. and 28 to 30 inch Hg vacuum for at about 20 hours.

Example 2 Original Preparation of p-Methoxybenzylthioether Acid Chloride(2)

With reference to Equation II, p-Methoxybenzylthioether acid (400 g,1.57 mol) was charged into a 5-L round bottomed flask equipped with athermocouple, a mechanical stirrer, a reflux condenser topped with N₂inlet, and a 0.5-L pressure equalizing addition funnel. Methylenechloride (1,600 g, 1,212 mL) was charged into the 5-L reaction flask.The clear solution was heated to 20 to 25° C. Methylene chloride (300 g)and oxalyl chloride (110 g, 78 mL) were charged into the 0.5-L pressureequalizing addition funnel. 350 mL of oxalyl chloride/methylene chloridesolution were added via the addition funnel while maintaining thereaction temperature at 20 to 30° C. The clear yellow solution wasstirred at 20 to 25° C. for a minimum of 30 minutes until bubblingsubsided. Addition of oxalyl chloride was repeated. 350 ml of oxalylchloride/methylene chloride solution were added via the pressureequalizing addition funnel to the reaction flask while maintainingreaction temperature at 20 to 30° C. (Addition time about 45 minutes).The reaction mixture was heated to about 32 to 38° C. The stirredsolution was held in this temperature range for a minimum of 1 hour. A 1mL sample was removed for HPLC analysis. The reaction was deemedcomplete when less than 3% by area of the methoxylbenzylthioether acidstarting material remained. The reaction was cooled to from 23 to 28° C.over a minimum of 5 minutes. The solution was transferred to a tared 3-Lround bottom flask. The reaction flask was rinsed to the 3-L flask with100 ml, 132 g methylene chloride. The reaction solution was concentratedsolution in vacuo, by rotary evaporator with bath temperature set at 33to 36° C. and a pressure of 25 to 28 inch Hg, until no volatilesremained. The final weight was 1,367 g and the net weight was 500.3 gofp-methoxybenzylthioether acid chloride. Distillate was discarded.

Example 3 Original Preparation of p-Methoxybenzylthioether Hydrazide (3)

With reference to Equation III, a 5-L Morton type round bottomed flaskwas equipped with a thermocouple, a mechanical stirrer, a refluxcondenser topped with N₂ inlet, and a 0.5-L pressure equalizing additionfunnel. p-methoxylbenzylthioether acid chloride was dissolved in 500 ml,660 g of methylene chloride. The solution was transferred into a 2-LErlenmeyer flask. 500 ml methylene chloride were added to make up atotal volume of 1,300 mL solution.

In a 5-L Morton-type round-bottomed flask (RBF), Morton Type, werecharged 2,400 g, 1,818 mL of methylene chloride and 256 g, 245 mL, @ 98%strength, 7.8 mol of anhydrous hydrazine. The mechanical stirrer speedwas set at 255-270 rpm. The cloudy slurry was cooled to −69 to −72° C.using dry ice/acetone.

The acid chloride solution was added drop wise to the 5-L reaction flaskvia the 0.5-L pressure equalizing addition funnel, maintaining areaction temperature of −68 to −72° C. It was important to adjust theaddition rate of methylbenzylthioether acid chloride solution to thestirred hydrazine/methylene chloride slurry at a rate that ensured areaction temperature of less than −67° C. Addition was complete afterabout 3 hours. The stirred reaction was held at −68 to −72° C. for aminimum of 30 min. A 1 mL sample was removed for HPLC analysis. Thesolution was warmed to room temperature (20 to 30° C.) by removing theice bath.

The reaction mixture was transferred into a tared 3-L round bottomflask. The reaction solution was concentrated in vacuo by rotaryevaporator. The bath temperature was set at 32 to 36° C. and pressure 25to 28 inch Hg. All volatiles were removed. The final weight was 1,490.7g and the net weight was 630 g of crude, solid p-methoxylbenzylthioetherhydrazide. The distillate was discarded.

Methanol (1,250 g, 1,580 mL) was added to crudep-methoxylbenzylthioether hydrazide solid and the heterogeneous mixturewas mixed in a 5-L round-bottom flask at 33 to 36° C. for minimum of 5minutes until a clear solution was obtained. The crudep-methoxylbenzylthioether hydrazide/methanol solution was transferredinto a 5-L reaction flask.

1,312 g of 4% sodium hydroxide/methanol solution were charged into the 5L reaction flask at 28 to 34° C. over 8 minutes. The clear mixture wasstirred at 33 to 36° C. for 20 minutes. A light precipitate formed.

The contents of the 5-L reaction flask were filtered on a 30-cm Buchnerfunnel with filter paper (Whatman #1). The (5-L) reaction flask wasrinsed with 200 mL, 158 g of methanol. The filtrate was transferred intoa tared 3-L round bottom flask. The reaction solution was concentratedin vacuo, by rotary evaporator. The bath temperature was set at 36 to40° C. and pressure 25 to 28 inch Hg. All volatiles were removed. Thefinal weight was 1,484 g and the net weight was 622.7 g of crudep-methoxybenzylthioether hydrazide solid. The distillate was discarded.The solid was dissolved in a 700 g, 530 mL of methylene chloride. Theheterogeneous mixture was mixed in the rotary evaporator (no vacuum), at33 to 36° C. for minimum of 10 minutes until clear solution wasobtained.

The solution was concentrated in vacuo by rotary evaporator. The bathtemperature was set at 36 to 40° C. with a pressure of 28 to 30 inch Hg.All volatiles were removed. The final weight was 1,494.2 g and the netweight was 632.5 g of crude p-methoxybenzylthioether hydrazide solid.The distillate was discarded. The solid was dissolved with 2,100 mL,2,772 g of methylene chloride. The heterogeneous mixture was mixed at 20to 25° C. for a minimum of 5 minutes until a clear solution wasobtained. 110 g of anhydrous magnesium sulfate was added to themethylene chloride solution, and the mixture was stirred for 1 hour.Using suction, the yellow mixture was filtered through a 15-cm Buchnerfunnel with filter paper (Whatman #1) into a 5-L reaction flask. Theflask and filter cake were rinsed with 500 mL, 660 g of methylenechloride. The filtrate was transferred into a tared 3-L round-bottomflask. The solution was concentrated in vacuo, by rotary evaporator. Thebath temperature was set at 32 to 35° C. with pressure of 20 to 25 inchHg. All volatiles were removed. The fmal weight was 1,090 g and the netweight was 643 g of crude p-methoxybenzylthioether hydrazide solid. Thedistillate was discarded.

The solid was dissolved with 400 mL, 528 g of methylene chloride. Theheterogeneous mixture was mixed at 35 to 40° C. for minimum of 5 minutesuntil a clear yellow solution was obtained. 4,260 mL, 3,040 g of etherwas charged to a 12-L round bottomed flask equipped with a thermocouple,a mechanical stirrer, an N₂ inlet, and a 2-L pressure equalizingaddition funnel. The ether was cooled in the 12-L flask to 0 to −10° C.using brine and ice. The yellow p-methoxybenzylthioetherhydrazide/methylene chloride solution (prepared above) was added viapressure equalizing addition funnel to the rapidly stirred 300-400 rpmether while maintaining the temperature at 0 to −10° C. 1,070 mL, 732 gof heptane was charged. The heterogeneous mixture was stirred at 0 to−5° C. for 20 minutes. The contents of the (12-L) reaction flask werefiltered through a 30-cm Buchner funnel with filter paper (Whatman #1).The filtrate was collected in a 4-L suction flask. The 5-L reactionflask was rinsed to the cake with 1,070 mL, 732 g of heptane. Thefiltrate was discarded. The filter cake was dried with suction for aminimum of 50 minutes until essentially no more filtrate was collected.The filter cake height was 15 mm. Filter cake weight was 429 g. The wetcake was transferred into a drying dish. The drying dish containing themethoxybenzylthioether acid was covered with clean filter paper. Theproduct was dried in a vacuum oven at 38 to 40° C. and 28 to 30 inch Hgvacuum for at least 18 hours.

Results for typical batch reactions using the methods of Examples 1-3are gathered in Table 2.

TABLE 2 TYPICAL BATCHES BEFORE PROCESS IMPROVEMENTS WERE ADDED CorrectedStrength of Bis-hydrazide hydrazide Hydrazide by-product Yield Batch No(%) (%) (%) 2.1 78.5 22.1 67.8 2.2 78.4 19.3 74.8

Example 4 Modified Preparation of p-Methoxybenzylthioether Hydrazide (3)

In order to reduce the level of the by-product,bis-methoxybenzylthioether hydrazide (6), the reaction parameter(s) thatinfluence the formation of this by-product in the isolated product,p-methoxybenzylthioether hydrazide were investigated. The procedure ofExample 3 was repeated. At −78° C. the hydrazine/CH₂Cl₂ solution is anunstirrable frozen mixture. Gummy lumps stuck to the reaction flaskwhile the stirring blade was spinning in the air. A solution ofthioether acid chloride/CH₂Cl₂ was added drop wise to this frozenmixture of hydrazine/CH₂Cl₂ (28% v/v), while keeping the temperature atabout −72° C. HPLC analysis at the end of the addition (temperature was−72° C.) showed little reaction. This was contrary to the expectationthat it is a fast reaction. This could be due to lack of proper mixingin the reaction. The largely unreacted reaction mixture was allowed towarm. When the temperature reached about −50° C., a stirrableheterogeneous mixture developed, followed by a rapid exotherm thatpushed the temperature instantly to −28° C. where the reaction colorchanged from yellow to off-white. This led to the postulation thatineffective mixing could lead to localized reaction that favored thegeneration of the bis-hydrazide. Warming to room temperature and work-upof the reaction as in Example 3 provided the bis-hydrazide as the majorproduct (82%, HPLC area %). This is much higher than the typicalundesired level of 20%. It was concluded that the solution of addedthioetheracid chloride (2)/CH₂Cl₂ did not mix effectively with thefrozen lumps of hydrazine.

Example 5 Temperature Effects on the Preparation ofp-Methoxybenzylthioether Hydrazide (3)

Example 5 repeated the same reaction of Example 4, but was carried at 0°C., rather than at about −72° C. A solution of thioether acidchloride/CH₂Cl₂ was added drop wise to a stirrable, homogenous solutionof hydrazine/CH₂Cl₂ (28% v/v). In this case, 39% (HPLC area %) of thebis-hydrazide formed. These conditions imply that lower temperature andstirring are factors that affect the formation of undesired by-product.The results of Examples 3.1 and 3.2 are gathered in Table 3.

TABLE 3 PREPARATION OF P-METHOXYBENZYLTHIOETHER HYDRAZIDE AS A FUNCTIONOF TEMPERATURE Hydrazine/ Bis- Example Hydrazine CH₂Cl₂ Temp hydrazideNo. (Eq.) (v/v) (%) (° C.) (%) Comments 3.1 5 28 ~−72 82 Poorly stirred3.2 5 28 ~0 39 Stirred slurry a. The acid chloride was added to thehydrazine/CH₂Cl₂ mixture at rate of 0.25 mL/min.

Example 6 Effect of Hydrazine Concentration on the Preparation ofp-Methoxybenzylthioether Hydrazide (3)

The effect of using a lower concentration of hydrazine at lowtemperature was then examined. The results are gathered in Table 4below. A stirrable, heterogeneous mixture of hydrazine/CH₂Cl₂ at −65 to−72° C. was prepared by diluting the hydrazine/CH₂Cl₂ to concentrationsof 5% and 19% vs 28% (v/v). Experiments 4.1 and 4.2 in Table 4 carriedout at hydrazine/CH₂Cl₂ concentrations of 19% and 5%, respectively,provided a stirrable, heterogeneous mixture that was reacted withthioether acid chloride to provide desired product with bis-hydrazideby-product levels of 3% and 5%, respectively. Repeating the samereaction using less hydrazine (Experiment 4.3 in Table 4 using 5 vs 10mol equivalents) generated only 3% of the bis-hydrazide. The typicalamount of hydrazine is 5 mol equivalents versus thioether acid. Doublingthe amount of hydrazine to 10 mol equivalents (Table 4: Experiments 4.1and 4.2) did not significantly affect the level of bis-hydrazide in thefinal product.

Adding the acid chloride at a faster rate (cf. experiments 4.4 and 4.5in Table 4, which used 1 vs 0.25 mL/min.) to the dilute hydrazine/CH₂Cl₂heterogeneous mixture (5% and 19%) generated bis-hydrazide at levels of3% and 9%, respectively. The drastic drop in the level of thebis-hydrazide (from 82% to 3%, cf. experiment 3.1 in Table 3 above andexperiment 4.4 in Table 4) could be attributed to one or more of thefollowing factors: Temperature, concentration, amount of hydrazine,addition rate and mixing. The 9% bis-hydrazide generated from Experiment4.5 in Table 4 could be caused by initial ineffective stirring.

TABLE 4 PREPARATION OF P-METHOXYBENZYLTHIOETHER HYDRAZIDE UNDER VARIOUSDILUTIONS OF HYDRAZINE/CH₂CL₂ Addition Hydrazine/ Bis- Exp. No.Hydrazine rate CH₂Cl₂ hydrazide (5 g Scale) (Eq.) (mL/min.) (v/v) (%)(%) Comments 4.1 10 0.25 19 4 Stirred slurry 4.2 10 0.25 5 5 Stirredslurry 4.3 5 0.25 5 3 Stirred slurry 4.4 5 1.0 5 3 Stirred slurry 4.5 51.0 19 9 Started as non-stirred slurry The reaction temperature duringthe addition was maintained at −68 to −73° C..

Example 7 Effect of Temperature on the Preparation ofp-Methoxybenzylthioether Hydrazide (3)

The effect of temperature on the level of bis-hydrazide in the productwas examined in Table 5. In experiments where the thioether acidchloride was added to a stirred mixture of hydrazine/CH₂Cl₂ (19% v/v) at−20 and −72° C. (Table 5: Experiment 5.1 and 5.2), bis-hydrazide wasgenerated at levels of 28% and 4%, respectively. Similar results wereobserved in experiments 5.3 and 5.4 (Table 5). The above resultsindicate a lower reaction temperature (˜−70° C.) is necessary to obtainlower levels (3-5%) of the bis-hydrazide.

TABLE 5 THE EFFECT OF TEMPERATURE ON THE LEVEL OF THE BIS-HYDRAZIDEFORMED Experiments^(a) Hydrazine/CH₂Cl₂ Temp Bis-hydrazide (5 g Scale)(v/v) (%) (° C.) (%) Comments 5.1 19 −20 28 stirred slurry 5.2 19 −72 4stirred slurry 5.3 5 −38 16 stirred slurry 5.4 5 −72 3 stirred slurry^(a)The above experiments were carried out using 5 eq. of hydrazine.

Example 8 Effect of Hydrazine Concentration on the Preparation ofp-Methoxybenzylthioether Hydrazide (3)

Examining the experiments in Table 4 revealed that an additiontemperature of around −70° C. and a concentration of hydrazine/CH₂Cl₂ ateither 19% or 5% (v/v) produced comparable results. This observation wasfurther examined in Table 6. In experiments 6.1, 6.2 and 6.4 in Table 6where the thioether acid chloride was added to the heterogeneous mixtureof hydrazine/CH₂Cl₂ at concentrations of 19%, 14% and 10%, thebis-hydrazide was generated at levels of 6%, 13% and 4%, respectively.The reaction volume, flask size and agitation speed were kept constant.The results showed that at 19%, the reaction was comparable to theresults at 10% concentration. The hydrazine/CH₂Cl₂ concentration of 14%was repeated at 30 g scale (Experiment 6.3 in Table 6) to provide thedesired product contaminated with only 3% bis-hydrazide. The higherlevel of bis-hydrazide in Experiment 6.2 is attributed to initial fastaddition of acid chloride that caused the reaction temperature to spiketo −57° C. before it was quickly adjusted.

TABLE 6 THE EFFECT OF HYDRAZINE/CH₂CL₂ CONCENTRATION ON THE LEVEL OF THEBIS-HYDRAZIDE FORMED Hydrazine/ Exp No. ^(a) CH₂Cl₂ Temp Bis-hydrazideComments (Scale) (v/v) (%) (° C.) (%) (Liq. vol/RBF size) 6.1 19 −68 to−70 6  20 mL/100 ml (5 g) 6.2 14 −57 to −65 13 (Initial fast add.) (4.2g)  20 mL/100 ml 6.3 14 −68 to −71 3 140 mL/500 mL (30 g) 6.4 10 −61 to−68 4  20 mL/100 ml (2.9 g) ^(a) the above experiments were carried outusing 5 equivalents of hydrazine

Example 9 Effect of Mixing Speed on Preparation ofp-Methoxybenzylthioether Hydrazide (3)

The effect of mixing was examined (Table 7). Experiments 7.2 and 7.1showed that faster mixing (400 rpm vs 200 rpm) produced lesserbis-hydrazide (22% vs 40%). The higher than usual level of bis-hydrazidein both experiments could be caused by ineffective mixing consideringthe initial liquid level (36 mL) of hydrazine/CH₂Cl₂ in the 50-mL flaskcompared to 20 mL liquid in 100-mL flask in Table 8. This means that inaddition to the speed of the mixing, the reactor geometry and liquidlevel must be considered.

TABLE 7 THE EFFECT OF MIXING ON THE LEVEL OF THE BIS-HYDRAZIDE FORMEDHydrazine/ Mixing Experiment CH₂Cl₂ Temp Bis-hydrazide Add rate rate (5g scale) (v/v) ( %) (° C.) (%) (mL/min.) (rpm) 7.1 19 −60 40 1 200 7.119 −65 22 1 400 a. The final liquid volume/reactor size was 72%.

Example 10 Scale-Up Effects on Preparation of p-MethoxybenzylthioetherHydrazide (3)

Preparing p-methoxybenzylthioether hydrazide at either concentration ofhydrazine/CH₂Cl₂ of 19% or 5% (v/v) produced comparable results. Whilethe 19% concentration of hydrazine/CH₂Cl₂ is usually a stirrable mixtureat −70° C., there is a risk that it could become a poorly mixed frozenmixture. If a manufacturing scale batch (400 g) were run at 5%concentration of hydrazine/CH₂Cl₂, a larger reactor (20-L Morton type)could be required. However, if the process is run at 14% concentrationof hydrazine/CH₂Cl₂, the reaction can be carried out using a glassreactor, 5-L Morton type round-bottomed flask. When the process was runat 14% v/v concentration of hydrazine/CH₂Cl₂ at 20 g scale (Experiment8.1 in Table 8) the isolated product, p-methoxybenzylthioetherhydrazide, was contaminated with 4.4% (HPLC area %) of by-product,bis-methoxybenzylthioether hydrazide. Repeating the above condition(concentration of hydrazine/CH₂Cl₂ was 14%) at manufacturing scale-upconditions (Experiment 8.2 in Table 8) produced p-methoxybenzylthioetherhydrazide contaminated with 4.2% (HPLC area %) of by-product,bis-methoxybenzylthioether hydrazide.

TABLE 8 PREPARATION OF P-METHOXYBENZYLTHIOETHER HYDRAZIDE ATHYDRAZINE/CH₂CL₂ CONCENTRATION WAS 14% Initial Liq. level/ Bis-hydrazideExp. No. Flask size reactor size HPLC area % (Scale) (mL) (%) (%)8.1^(a)  250 39^(c) 4.4 (20 g) 8.2^(b) 5000 39^(c) 4.2 (400 g) (Morton)^(a)The concentration of hydrazme/CH₂Cl₂ mixture is 14% (v/v) ^(b)Theconcentration of hydrazine/CH₂Cl₂ mixture is 14% (v/v). ^(c)The initialvolume reactor size = 39%. Final liquid volume/reactor size = 64%.

The original process (Table 9, experiment 9.1), provided the product,p-methoxybenzylthioether hydrazide, in 78.4% strength that contained19.3% of by-product, bis-hydrazide. The modified process in Experiment9.2, Table 9, used continuous addition of a methoxybenzylthioether acidchloride solution to a more dilute and stirrable hydrazine/methylenechloride heterogeneous mixture (concentration was 14%). The product,p-methoxybenzylthioether hydrazide, was prepared in 91.1% strength thatcontained 4.7% of by-product, bis-hydrazide.

TABLE 9 COMPARISON BETWEEN THE MODIFIED PROCESS AND THE PREVIOUS PROCESSFOR THE PREPARATION OF P-METHOXYBENZYLTHIOETHER HYDRAZIDE Mixing Flaskp-Methoxybenzylthioether Process speed size hydrazide Bis-hydrazide(Scale) (RPM) (mL) (Strength %) (%) 9.1^(a) nd 5,000^(c) 78.4 19.3 400 g(Morton) 9.2^(b) 270 5,000^(d) 91.1 4.7 400 g (Morton) ^(a)Theconcentration of hydrazine/CH₂Cl₂ mixture is 28% (v/v) ^(b)Theconcentration of hydrazine/CH₂Cl₂ mixture is 14% (v/v). ^(c)The Initialvolume/reactor size was 21%. Final liquid volume/reactor size was 47%.^(d)The Initial volume/reactor size was 39%. Final liquid volume/reactorsize was 64%.

The following conditions ensure a low level of the bis-hydrazideby-product is formed and are recommended for the manufacturingofp-methoxybenzylthioether hydrazide. These reaction conditions areconsidered aspects of certain embodiments of the present invention:

1. Addition rate of methoxybenzylthio acid chloride solution tohydrazine/CH₂Cl₂ mixture adjusted to maintain reaction temperature of−68 to −75° C.2. Effective mixing (initial liquid volume of 30 to 40% liquid volumeversus reactor size) at high mixing speed (300 to 400 rpm). Mixingmaintained at speed of 260-270 rpm in 5 L Morton-type flask.3. Stirrable, uniform mixture of hydrazine/CH₂Cl₂ (concentration was 5to 19%, v/v). In one example the ratio of hydrazine/CH₂Cl₂ was 14%, v/v.4. Initial liquid volume of 30 to 40% liquid volume versus reactor size.

The following examples 11-15 combine to illustrate a preferredembodiment of the invention.

Example 11 Modified Preparation of the p-Methoxybenzylthioether AcidIntermediate (1)

A 5-L reaction flask with a condenser, N₂ inlet, stirrer, andtemperature probe/controller was set up. Piperidine (0.402 kg) wascharged into the vessel under a N₂ atmosphere. 3,3-dimethylacrylic acid(0.215 kg) was added portion wise with stirring followed byp-methoxybenzylthiol (0.358 kg). The reaction mixture was heatedgradually to 82 to 88° C. over a minimum of 15 minutes and that reactiontemperature was maintained until an exotherm was observed. Thetemperature was not allowed to exceed 95° C. When the exotherm wascomplete, heating was continued to 92 to 98° C. and maintained for aminimum of 15 hours.

Three liters of 3 M aqueous HCl were prepared. The heating mantle wasremoved and the reaction mixture was allowed to cool to 70 to 75° C. 1.9L of the HCl solution was slowly added. Cooling was continued with awater bath until the flask contents reached a temperature of 20 to 30°C. CH₂Cl₂ (1.64 kg) was added and the flask contents were stirred for aminimum of 5 minutes. The pH was checked and adjusted to <2 as neededusing the HCl solution. The reaction mixture was transferred to aseparatory funnel, the phases were allowed to separate, and the lowerorganic product layer was drained back into the reaction flask. Theupper aqueous layer was transferred to a separate flask. The remainingHCl solution was added to the organic phase and stirred for a minimum of5 minutes. The pH was checked and adjusted to <2 as needed with fresh 3M HCl solution.

The contents of the reaction flask were returned to the separatoryfunnel and the layers were allowed to separate for a minimum of fiveminutes. The lower organic phase was drained to a clean Erlenmeyer flaskand the aqueous layer was drained to the reaction flask. The aqueousphase from the previous extraction was added to the reaction flask aswell as CH₂Cl₂ (0.400 kg). The reaction was stirred for a minimum of 5minutes, then the contents of the reaction flask were transferred to theseparatory funnel and the layers were allowed to separate for a minimumof five minutes. The lower organic product phase was combined with theprevious organic product phase and transferred to the reaction flask.The combined aqueous phases were discarded as waste. Water (1.00 kg) wascharged to the reaction flask and stirred for a minimum of 5 minutes.The mixture was transferred to the separatory funnel and the phases wereallowed to separate for a minimum of five minutes. The lower productorganic phase was drained to a clean Erlenmeyer flask. The aqueous phasewas discarded as waste. The organic product solution was dried overanhydrous MgSO₄, then suction filtered into a 5-L 4-necked flask.

The Erlenmeyer flask and filter cake were rinsed into the 5-L 4-neckedflask with CH₂Cl₂ (0.350 kg). CH₂Cl₂ was distilled off to a pot volumeof 900±50 mL. The temperature of the concentrate was adjusted to 15 to20° C. and then precipitated by adding heptane (1.67 kg). The mixturewas cooled to about 5° C. and stirred for a minimum of 30 minutes, thenthe batch was suction filtered and the product cake rinsed with heptane(2×0.272 kg). The product was sampled for loss on drying analysis (LOD)and the filtrate sampled for Solids Content Projection. If the filtratecontains >0.110 kg, then it is concentrated and treated with heptane toprecipitate a second crop of the product as before. The damp productcake(s) were weighed and the dry weight calculated using the LOD data.The dry mass of the product was equivalent to a constant A (kg). Thedamp product was returned to the 5-L flask and dissolved in CH₂Cl₂(1.76×A kg minimum, 2.20×A kg maximum). Heptane was added (4.56×A kg)slowly, initiating precipitation. The slurry was cooled to 0 to 5° C.then aged for a minimum of 30 minutes. The batch was suction filteredand the product cake rinsed with heptane (2 portions, each 0.272 kg).Filtration was continued until filtrate flow essentially stopped. Thewet product cake was transferred to a tared dish(es), a weight wasobtained, then the filtrates were transferred to an appropriate wastecontainer. The cake was dried in a vacuum oven at not more than 38° C.until a loss on drying LOD spec. of <1.0% was met. The sample wassubmitted for analysis.

Example 12A Preparation of the p-Methoxybenzylthioether Acid Chloride(2)

A 5-L reaction flask equipped with a condenser, water scrubber,temperature probe, 1-L addition funnel, N₂ inlet, and stirrer was setup. CH₂Cl₂ (1.6 kg) was charged under a N₂ atmosphere followed byp-methoxybenzylthioether acid (0.400 kg) with stirring. A solution ofoxalyl chloride (0.220 kg) and CH₂Cl₂ (0.600 kg) was prepared in theaddition funnel. About half of the oxalyl chloride solution was addedwhile maintaining a temperature range of 20 to 30° C. (exothermic!).CO₂/CO evolution was observed while stirring for a minimum of 30minutes, then the remaining oxalyl chloride solution was added, whilemaintaining the temperature between 20 and 30° C. The reaction wasstirred until gas evolution diminished (about 30 min), then the mixturewas heated to 33 to 38° C. This temperature was maintained for about 60minutes until gas evolution diminished. The reaction was sampled, andHPLC was used to determine the amount of acid remaining. The reactionwas judged complete when the amount of starting material was not morethan 5%. If the reaction was not complete, then stirring was continuedat 33 to 38° C. for an additional hour, then sampled and tested again.The heating mantle was removed and the reaction mixture was allowed tocool to 20 to 30° C. The mixture was transferred to a 3-L single-neckedflask, then rinsed in with CH₂Cl₂. The batch was concentrated on arotary evaporator until most of the volatiles were removed.

Example 12B Preparation of the p-Methoxybenzylthioether HydrazideIntermediate (3)

A 5-L 4-necked Morton type reaction flask with condenser, N₂ inlet,thermocouple, stirrer, and 2-L addition funnel was set up. CH₂Cl₂ (2.40kg) was charged under a N₂ atmosphere and cooled to −75 to −65° C.Anhydrous hydrazine (0.252 kg) was charged to give a uniform slurry ofhydrazine ice, without formation of hydrazine crystals on the sidewallsof the flask. The thioether acid chloride solution was transferred tothe addition funnel, rinsing in CH₂Cl₂ as necessary to give a solutionvolume of 1.30 L.

The acid chloride solution was added drop wise at a steady rate over aminimum of three hours while maintaining the temperature between −65 to−75° C. (preferably between −70 to −75° C.). After the addition wascomplete, the reaction was stirred at −65 to −75° C. for a minimum of 30minutes. The batch was warmed to 20 to 25° C. The reaction was sampledby HPLC and was judged complete if the amount of remaining acid chloridewas not more than 5%. If the reaction was incomplete at this point,stirring was continued at 20 to 25° C. for a minimum of one hour, thensampled again. The batch in a 3-L flask was concentrated on the rotaryevaporator. The batch was rinsed into the flask with CH₂Cl₂ as required.The concentrate was diluted with MeOH (1.25 kg) and transferred to a 5-Lreaction flask equipped with stirrer, thermocouple, and N₂ inlet, rinsedin with MeOH as needed.

A solution of NaOH (0.0640 kg) in MeOH (1.25 kg) was added under a N₂atmosphere and stirred for a minimum of 20 minutes. The batch wasclarified by suction filtration and rinsed as required with MeOH. Thefiltrate was transferred to a tared 3-L flask (rinsed in with MeOH asrequired) and concentrated on the rotary evaporator until all of thevolatiles were removed, then continued in vacuo for a minimum of 30minutes. Application of vacuum was discontinued and CH₂Cl₂ (0.704 kg)was added. Rotation was continued to effect dissolution of theconcentrate, then the application of vacuum was resumed and concentratedto a solid residue. The weight of the residue was obtained and theresidue was transferred to a separatory funnel using CH₂Cl₂ (2.84 kg).The mixture was agitated to give a solution.

The CH₂Cl₂ solution was washed with two portions of water (1.00 kgeach). Anhydrous MgSO₄ (0.300 to 0.420 kg) was added to the CH₂Cl₂solution and swirled for about fifteen minutes until the solution wasclear. The batch was suction filtered, rinsing with CH₂Cl₂ as required.The filtrate was transferred to a tared 3-L flask and concentrated on arotary evaporator to a solid residue. The weight of the residue wasobtained, and it was dissolved in CH₂Cl₂ (not less than 0.532 kg). Thesolution was transferred to a 1-L addition funnel that was attached to a12-L 4-necked reaction flask equipped with a stirrer, thermocouple, andN₂ inlet. Ether (3.04 kg) was charged to the flask under a N₂atmosphere. The ether was cooled to 0 to −10° C.

The p-methoxybenzylthioether hydrazide solution was added to the rapidlystirring ether solvent while maintaining the temperature between −10 and0° C. Additional solution was rinsed into the flask using CH₂Cl₂ (0.0660kg). Heptane (0.732 kg) was charged to the addition funnel and was addedslowly to the thin slurry, again maintaining the same temperature range.The resulting slurry was stirred at the same temperature for a minimumof 60 minutes. The batch was suction filtered through paper. The productcake was rinsed with heptane (2 portions, each 0.366 kg) and suctiondried until a wet cake formed. The wet cake was transferred to tareddishes and the weight of the cake was obtained. The cake was dried in avacuum oven (not more than 38° C.) until LOD was not more than 2.0%. Theweight of the dried p-methoxybenzylthioether hydrazide was obtained andsamples were submitted for testing.

Example 13 Preparation of Thiol-deprotected Intermediate (4)

Dowex SRB OH anionic exchange resin was prepared by adding 2.4 kg of theresin to a large Büchner funnel and washing with water (4 portions, each2.40 kg) then MeOH (4 portions, each 1.92 kg). The resin was coveredwith water in a beaker and soaked for a minimum of one hour, then thewater was filtered off. The resin was transferred to an appropriatestorage container. A 5-L reaction flask equipped with a stirrer,thermocouple, N₂ inlet, and a 250 mL addition funnel was set up.Trifluoroacetic acid (2.80 kg) was charged under a N₂ atmosphere andcooled to 5 to 10° C. Thioether hydrazide (0.380 kg) was added portionwise (exothermic!), while maintaining the temperature between 5 and 15°C. The solution was cooled to 0 to 5° C.

Trifluoromethanesulfonic acid (0.243 kg) was charged to the additionfunnel and added to the reaction mixture, while maintaining thetemperature between 0 to 10° C. After the addition was complete, anisole(0.0152 kg) was added. The reaction mixture was stirred at 10 to 15° C.for a minimum of two hours or until the reaction color was deep red anddid not change further. The reaction was sampled and tested by TLC andjudged complete if the reaction mixture contained not more than 4%starting material. A 12-L reaction flask equipped with a stirrer, N₂inlet, and 2-L addition funnel was set up. MeOH (3.01 kg) was charged.The vessel was cooled to 0 to 5° C. under a N₂ atmosphere. The reactionmixture was transferred to the addition funnel and then added to thechilled MeOH at a moderate rate, maintaining a reaction temperature of 0to 5° C. A white precipitate formed. The reaction flask was rinsed intothe addition funnel with additional MeOH (0.0790 kg). The white slurrywas stirred for about 15 minutes at 0 to 5° C. The batch was suctionfiltered through paper and the product cake was rinsed with MeOH (2portions, each 0.600 kg). After filtrate flow through the cake hadessentially stopped, the filtrate was transferred to a tared 3-L flask(rinsed in with MeOH as required) and concentrated on the rotaryevaporator to a semi-solid residue.

The residue was redissolved in MeOH (0.600 kg) and concentrated again asbefore. The residue was redissolved in CH₂Cl₂ (0.600 kg), concentratedagain as before and the weight of the residue was obtained. The residuewas dissolved in water (1.52 kg) and the solution was transferred to a6-L separatory funnel. The residue was washed with CH₂Cl₂ (threeportions, each 0.927 kg). The combined organic phases were transferredto an appropriate waste container. The aqueous phase containing theproduct was transferred to a beaker and the pH was adjusted to 6.5 to7.5 pH units by addition of the swelled resin. The pH was adjusted withtrifluoroacetic acid as necessary. Once the desired pH range wasachieved, the slurry was stirred for about 30 minutes, and the pH waschecked again, and adjusted if necessary. The batch was suction filteredand the resin was rinsed with water (2 portions, each 0.600 kg). 37% HClsolution (0.160 kg) was added. The pH was measured to ensure that it was<1.5. Additional HCl was added if necessary. The aqueous solution wastransferred to a tared 3-L flask (rinsed in with water as required) andconcentrated to a solid residue on the rotary evaporator. The weight ofthe residue was obtained. The residue was dissolved in absolute EtOH(1.20 kg) and concentrated again. The residue was re-dissolved inabsolute EtOH (1.65 to 2.85 kg) and heated to 50 to 65° C. The warmsolution was suction filtered. The filtrate was transferred to a tared3-L flask and the ethanol solution concentrated on a rotary evaporatoruntil distillation essentially ceased. EtOAc (8.21 kg) was charged inportions and concentrated as before. A fourth portion of EtOAc (2.74 kg)was charged and cooled to 20 to 25° C. Reaction was stirred for about 15minutes. The batch was suction filtered and rinsed with EtOAc (2portions, each 0.135 kg). Suction was continued until the filtrate flowessentially stopped. The cake was suction dried for about 60 minutes.The filtrate was discarded.

The weight of the damp product cake was obtained and that mass (kg) wasused in subsequent calculations as a constant B. The product wastransferred to a 12-L reaction flask equipped with a condenser, stirrer,N₂ inlet, and thermocouple. EtOAc (45.1×B kg) was charged under N₂ andthe slurry heated to 48 to 53° C. with stirring. Heating wasdiscontinued upon reaching 50° C. The heating mantle was removed and theslurry was cooled to 20 to 25° C. The batch was suction filtered andrinsed with EtOAc (2 portions, each 0.270 kg). Suction was continueduntil the filtrate flow essentially stopped. The filter cake was driedin a vacuum oven at not more than 38° C. for a minimum of 12 hours. Thefilter cake weight was obtained and the product mass (kg), equivalent toa constant C, was used in subsequent calculations.

Example 14 Preparation of Freebase Hydrazide (5)

The crude hydrochloride salt product (4) was mixed with water (20.0×Ckg) in a 12-L 4-necked reaction flask and stirred to give a solution.The pH of the solution was adjusted with the treated resin until a rangeof 6.5 to 7.5 pH units was achieved. The batch was stirred for about 15minutes, then the pH again was checked again and adjusted as needed toobtain a value of 6.5 to 7.5 pH units. The batch was suction filteredand rinsed with water (3.00×C kg), then with absolute EtOH (2 portions,each 5.30×C kg). Suction filtration was continued until filtrate flowessentially stopped. The product filtrate was transferred to a tared 3-Lflask and rinsed in with absolute EtOH as required. The batch wasconcentrated on the rotary evaporator until distillation essentiallystopped. The product residue was re-dissolved in absolute EtOH (1.58×Ckg) and concentrated as before. The product residue was re-dissolved inanhydrous ether (2.57×C kg) and concentrated as before. Oncedistillation essentially stopped, drying was continued with high vacuumand evaporation continued for a minimum of two hours. The net weight ofthe residue was a constant D. The concentrate was transferred to a 12-Lreaction flask equipped with a stirrer assembly, temperature probe, andnitrogen inlet using CH₂Cl₂ (66.3×D kg). The mixture was stirred at 15to 30° C. for a minimum of 30 minutes. The batch was filtered,collecting the filtrates in a second 12-L reaction flask. The firstflask was rinsed to the second flask through the filter using CH₂Cl₂(3.98×D kg). The second flask containing the batch was equipped with astirrer assembly, temperature probe, and nitrogen inlet. Silica gel(0.700×D kg) was charged with stirring and the stirring continued forabout 30 minutes. The batch was suction filtered and the silica waswashed with CH₂Cl₂ (2 portions, each 3.98×D kg), collecting the combinedfiltrates in a 10-L suction flask. The batch was concentrated on arotary evaporator into a 1-L flask at about 30° C. The 10-L flask wasrinsed to the evaporator as required with CH₂Cl₂. Distillation wascontinued until it essentially stopped. The rotary evaporator wasswitched to a high vacuum source and evaporation continued about threehours at 35 to 40° C.

The product oil crystallized by adjusting the rotary evaporator bath to0 to 5° C. while rotating the flask at the maximum rate. Evaporation wascontinued for about 30 minutes after the product solidified. The freebase product (5) was sampled and tested for residual methylene chlorideand dried until the test for residual solvent was acceptable. The finalweight was obtained, the product packaged in amber glass bottles withcaps having inert liners, and samples submitted for testing.

Yield

The overall yield limit for the 5-step process is not less than 33% oftheory (0.105 kg) and the difference between the highest and lowestyield must be not greater than 15%. The limit for the former process was33 to 43% of theory; however, it is expected that the reduction inby-product formation afforded by the increase in methylene chloride inthe hydrazide formation reaction will cause an increased yield. Theactual yields in the validation batches will be used to define a yieldrange for production batches.

Example 15 Final Purification of 3-Methyl-3-Mercaptobutanoic AcidHydrazide, Cl-332258 (DMH Linker)

Methylene chloride (1,000 mL, 1,325 g) was charged to a 2 L, 4-neckedreaction flask equipped with a mechanical stirrer, N₂ inlet, refluxcondenser, and temperature control device. DMH linker (20 g) was chargedto a reaction flask. Under N₂, the slurry was stirred at 20±3° C. for aminimum of 30 minutes. The resulting cloudy solution of DMH linker wasfiltered through a 350 mL, medium sintered glass Buchner funnel. Thefiltrate was collected in a clean 2 L, 4-necked round bottom flaskequipped with a mechanical stirrer, N₂ inlet, reflux condenser, andtemperature control device. The reaction flask was rinsed to the cleanreaction flask with 20 mL, 26.5 g methylene chloride. Silica gel (20 g)was charged to the solution in the reaction flask while maintainingtemperature at 15-25° C. The slurry was stirred under N₂ at 20±3° C. fora minimum of 30 minutes. The heterogeneous mixture was filtered througha (350 mL, medium) sintered glass Buchner funnel. The filtrate wascollected in a clean 2 L single necked round bottom flask. The reactionflask was rinsed to the filter cake with methylene chloride (50 mL, 66.3g), collecting filtrate in single necked flask. The filtrate wasconcentrated to dryness using a rotary evaporator (bath=35±5° C.) and amechanical water aspirator 15-30 mm Hg, followed by high vacuum (7-10 mmHg). The resulting white solid was cooled to 0-5° C. and dried underhigh vacuum at 7 mm Hg for 2 hours. n-Heptane (100 mL, 68.4 g) wascharged to the hard solid and stirred at room temperature for a minimumof 10 minutes until a uniform suspension was obtained.

The product was isolated by suction filtration through filter paper (#1Whatman) on a 15 cm Buchner funnel. The 2 L flask was rinsed to thefiltercake with the n-heptane mother liquors, followed by 2×50 mL,2×34.2 g n-heptane washes. The filtercake was dried with suction at roomtemperature for a minimum of 5 minutes. The filtercake was transferredto an amber bottle placed in a vacuum desiccator and the damp cake wasdried 14.34 g to constant weight in vacuo (<10 mm Hg) at 20-25° C. for 3hours. Yield: 14.21 g. 75.9%, theoretical yield: 18.7 g.

Tentative Test Method Limit Found Strength HPLC, L18284-154 95% 102.8%Purity HPLC, L18284-148  5%  1.34% Largest Single Imp. HPLC, L18284-148 5%  1.03% Melting Point USP TBD 51.5-52° C.

1-33. (canceled)
 34. A method of preparing a hydrazide from hydrazineand an acyl halide comprising the steps of: (a) preparing a slurrycomprising hydrazine and an inert solvent; and (b) adding an acyl halidecontinuously to said slurry, wherein the acyl halide further comprises aprotected thiol.
 35. The method according to claim 34 wherein the acylhalide is added substantially drop wise to the slurry.
 36. The methodaccording to claim 34 wherein the acyl halide comprises a benzylthioether.
 37. The method according to claim 34 wherein the acyl has thestructure:

wherein P is a thiol protecting group; R₁ and R₂ are each selected fromthe group consisting of C₁-C₅ alkyl; X is a halide; and L is an alkylenelinker.
 38. The method according to claim 37 wherein L is —CH₂—.
 39. Themethod according to claim 37 wherein R₁ and R₂ are each independentlymethyl.
 40. The method according to claim 37 wherein P is a benzylgroup, optionally substituted on the phenyl ring.
 41. The methodaccording to claim 40 wherein P is p-methoxybenzyl group.
 42. The methodaccording to claim 34 wherein the acyl halide has the structure:

wherein X is a halide.
 43. The method according to claim 34 wherein theinert solvent is methylene chloride.
 44. The method according to claim34 wherein said hydrazide product has the structure:

wherein P is a thiol protecting group; R₁ and R₂ are each selected formthe group consisting of C₁-C₅ alkyl; and L is an alkylene linker. 45.The method according to claim 44 wherein L is —CH₂—.
 46. The methodaccording to claim 44 wherein R₁ and R₂ are each independently methyl.47. The method according to claim 44 wherein P is a benzyl group,optionally substituted on the phenyl ring.
 48. The method according toclaim 47 wherein P is p-methoxybenzyl group.
 49. The method according toclaim 44 wherein the hydrazide product is

or a salt thereof.
 50. The method according to claim 34 wherein thehydrazide contains less than 5% of a bis-hydrazide by-product having thestructure:

wherein R and R′ are optionally substituted alkyl, heteroalkyl, orheteroalkaryl groups.
 51. The method according to claim 50 wherein R andR′ are

wherein P is a thiol protecting group; R₁ and R₂ are each selected formthe group consisting of C₁-C₅ alkyl; and L is an alkylene linker. 52.The method according to claim 51 wherein L is —CH₂—.
 53. The methodaccording to claim 52 wherein R₁ and R₂ are each independently methyl.54. The method according to claim 51 wherein P is a benzyl group,optionally substituted on the phenyl ring.
 55. The method according toclaim 54 wherein P is p-methoxybenzyl group.
 56. The method according toclaim 55 wherein the bis-hydrazide by-product has a structure:


57. The method according to claim 34 wherein the continuous addition ofacid halide solution is adjusted to maintain reaction temperature ofabout −68° C. to about −75° C.
 58. A method of preparing3-methyl-3-mercaptobutanoic acid hydrazide comprising the step ofpreparing a hydrazide according to the method of claim
 48. 59. A methodof preparing an immunoconjugate of a member of the family ofcalicheamicins with a monoclonal antibody as carrier, which comprisespreparing 3-methyl-3-mercaptobutanoic acid hydrazide according to themethod of claim 58 and using the 3-methyl-3-mercaptobutanoic acidhydrazide for preparing said immunoconjugate.
 60. The method accordingto claim 59 wherein 3-methyl-3-mercaptobutanoic acid hydrazide is usedas a linker to make gemtuzumab ozogamicin or inotuzumab ozogamicin. 61.A method of preparing gemtuzumab ozogamicin comprising the steps ofpreparing the linker 3-methyl-3-mercaptobutanoic acid hydrazideaccording to the method of claim 58 and using said linker to linkcalicheamicin to the monoclonal antibody gemtuzumab.